Transcript CMC issues in production of therapeutic biologic

Working with FDA:
Biological Products and Clinical Development
Chemistry, Manufacturing and
Control Issues in Production of
Therapeutic Biologic Protein Products
Ingrid Markovic, Ph.D., Biologist
Laboratory of Biochemistry, Division of Therapeutic Proteins
Office of Biotechnology Products, Office of Pharmaceutical Science
Center for Drug Evaluation and Research
FDA
U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES
NATIONAL INSTITUTES OF HEALTH
OBP/OPS/CDER
Parallel Office to ONDQA, which also reviews proteins (e.g., insulin, HGH, etc.)
(courtesy of Dr. S. Kozlowski)
Working with FDA: Biological Products and Clinical Development
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Majority of Biotech Products Use Living
Cells to Produce a Protein Product
 Insert gene encoding the
protein of interest
 Cells require proper
conditions for optimal growth
(temp, pH, oxygen, feeds,
etc.)
 Culture and fermentation can
take weeks
 Complex Purification Steps
 Safe product with desired
potency
Bar Charts, Inc. 2003
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Protein Therapeutics
 Protein Therapeutics are licensed through
Biologics License Application (BLA) under
provisions of both Public Health Service (PHS)
and Food Drug & Cosmetic (FD&C) Acts
 Protein therapeutics are also regulated
through New Drug Application (NDA) under
provisions of FD&C Act (e.g., insulin & HGH)
 BLA under PHS act lacks an abbreviated
pathway for follow-on biologics or biosimilars
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How are Protein Therapeutics different
from Small Molecule Drugs?
 Contain intrinsic infectious agents
 Aseptic techniques required during production
(terminal heat or gamma sterilization rarely
applied)
 Usually have heterogeneous composition
• Numerous process and product-related
impurities
• Change in the manufacturing process can
cause change in product composition
 Exact structure may be unknown (e.g., all
possible variants often not fully characterized)
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Structure of Small Molecule vs.
Protein Drugs
Proteins have expected:
 Size, charge,
hydrophobicity
 Correct folding (S-S
bonds)
 Subunits
 Glycosylation
 Bioactivity
Statin
~400 Da
*
x
& Unexpected:
 Aggregation (side effects)
 Incorrect folding
 Amino acid modifications
– ox, deam, cys
Therapeutic protein ~5,000 - 300,000 Da
 Truncation, proteolysis
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Manufacturing Process
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Components of the
Manufacturing Process
 Expression vector (plasmid)
 Cell banking system
• Master Cell Bank (MCB)
• Working Cell Bank (WCB)
• End of Production Cells (EOP)
 Drug substance manufacturing and release
 Drug product formulation and release
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Expression Vector and Cell Banking System
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Source Materials
Bacteria
Mycoplasma
Fungi
Mice
Humans
Mammalian cell-culture
Yeast
Bacteria
Viruses
TSE
agents
Transgenics
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Expression Vectors (Plasmids)
 Used for transfer of genes from one organism to another
 Used for production of large amounts of protein
 Description of origin of the construct
 Plasmid mapping (e.g., restriction sites, integration sites,
promoter, copy number etc.) and stability
 Sequencing of gene of interest
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MCB and WCB
 A working cell bank
(WCB) is derived from the
master cell bank (MCB)
and is used to initiate a
production batch
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Characterization of Cell Banks
Test
Viability
Identity
Purity
Stability
*Karyology
*Tumorigenicity
MCB
X
X
X
X
X
X
WCB
X
X
X
EPC
X
X
X
X
X
*dependent upon cell substrate and manufacturing process
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Characterization of Cell Banks (cont.)
Test
Sterility (bacterial & fungal cont)
Mycoplasma
MCB
X
X
WCB
X
X
EPC
X
X
(cultivable/noncultivable)
Adventitious viruses
X
X
in vitro – cell lines
in vivo – mice, guinea pigs, eggs
Species-Specific
X
(MAP, HAP, RAP)
Retrovirus
X
X
(TEM, RT, infectivity)
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Sources of Adventitious Agents
 Cell Substrate
• Endogenous viruses
• Exogenous microbial contamination
• Source material screening:
– Human (HIV, HBV, HCV, CJD, etc.)
– Animal (TSE sources, species-specific viruses)
 Raw Materials
• Cell culture reagents (animal and non-animal
derived)
 Environment
• Water
• Air
• Humans/technicians
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Viral Clearance for Phase 1 IND
 Demonstration of viral clearance may be required. Exceptions:
certain source materials (e.g., E. coli, yeast) or in the event of
unmet medical need
 Perform small scale clearance study that mimics the clinical
purification process
• Spike Drug Substance with a model virus to demonstrate
viral removal by several logs beyond the potential load
• CHO cell substrate – demonstrate retroviral clearance
• Human cell substrate - demonstrate clearance of enveloped
and non-enveloped viruses (e.g., parvoviruses)
• Design the process upfront to adequately assess potential
risks
 Two orthogonal robust steps (e.g., low pH, nano-filtration,
solvent/detergent treatment, heat) typically included in the
purification process
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Production and Purification
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Downstream Processing
Upstream cell culture & fermentation
Isolation/Capture of protein
Purification
Drug substance
Formulation
Drug Product
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Fermentation Process
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Purification Process
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Drug Substance and Drug Product
Characterization
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Proteins Can be Heterogeneous
Mixtures
pyro-E
pyro-E
O
O
D
D
D
D
O
O
G
G
D
D
Pyro-Glu (2)

Deamidation (3 x 2)
 Methionine oxidation
(2 x 2)

Glycation (2 x 2)

High mannose, G0,
G1, G1, G2 (5)

Sialylation (5)
G
G
K
(9600)2≈

K
 C-term Lys (2)
108
(Courtesy of Dr. S. Kozlowski)
2 x 6 x 4 x 4 x 5 x 5 x 2 = 9600
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Drug Substance Characterization
 Drug Substance should be positive for identity and have
specified criteria for purity, potency and microbial
contamination
 Acceptance criteria for release and stability attributes
should be established
• Often broader early in the development and subject
to revisions (e.g., narrowed down) as manufacturing
process develops
 Results from release and stability testing should be
provided in the IND
 Raw data supporting Drug Substance characterization
should be provided in the IND
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Drug Substance Characterization
(cont.)
 Safety
• Ensured by the specified limits for bioburden and endotoxin, misc.
process-related contaminants
 Purity & Characterization
• Assesses capability of purification process to remove processrelated impurities (e.g., endogenous viruses, host-cell proteins,
DNA, leachables, anti-foam, antibiotics, toxins, solvents, heavy
metals, etc.)
• Product-related impurities (e.g., aggregates, breakdown
products, product variants due to: oxidation, deamidation,
denaturation, loss of C-term Lys in MAbs etc.)
• Product substances (product variants that are active)
 Identity
• Unique for protein of interest, especially relevant for closely
related proteins manufactured in the same facility
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Drug Substance Characterization
(cont.)
 Potency
• Required to assess biological activity of the product
• Assay should be relevant for protein mechanism of action
• For MAb or Fc fusion proteins - a binding assay may be
sufficient for early development, but a functional assay
relevant for the mechanism of action should be developed
• If mechanism of action unknown - multiple bioactivities plus
elucidating higher order structure may be required
 Strength
• Protein content
 Stability
• Drug Substance stability should be demonstrated with
appropriate stability-indicating assays
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Drug Substance Characterization –
Methodology
 Safety
• LAL test, rabbit pyrogen test, bacterial culture methods
 Purity & Characterization including but not limited to:
•
•
•
•
•
Reversed-phase HPLC, Peptide mapping, MS
SDS-PAGE, Western analysis, capillary electrophoresis
SEC, AUC, FFF, light scattering
Ion Exchange Chromatography
Carbohydrate analysis (capillary electrophoresis, HPAEC = high-pH
anion-exchange chromatography, IEF for sialic acid)
 Identity
• N-terminal sequencing
• Peptide mapping
• Immunoassays (ELISA, Western blotting)
 Potency
• Animal-based assays, cell-based assays, reporter gene, biochemical
(e.g., enzyme activity)
 Protein content
• RIA, ELISA, UV absorbance, Bradford
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Drug Product Characterization
 Safety
• Final Drug Product for injection should be sterile
• Within specified limits for endotoxin
• Immunogenicity should be screened and monitored
– Successfully reduced in MAb by replacing murine
with human sequences
 Purity & Characterization
• Product and process-related impurities & productrelated substances should be within specified limits
 Identity
• Unique for protein of interest, especially relevant for
closely related proteins manufactured in the same
facility
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Drug Product Characterization (cont.)
 Potency
• Assay should be relevant for protein mechanism of action
• For MAb or Fc fusion proteins, a binding assay may be
sufficient for early development, but a functional assay relevant
for the mechanism of action should be developed
• If mechanism of action unknown - multiple bioactivities plus
elucidation of higher order structure may be required
 Strength
• Protein content
 Stability
• Drug Product should maintain stability for the duration of the
clinical trial
 Container closure compatibility
• Primary function - barrier to microbial ingress
• Extractables/Leachables studies – requirement for licensure
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Extractables
 Migrate from a c/c system and/or other
packaging components in DP vehicle or solvent
under extreme T°C and time conditions
exaggerated conditions
 Helpful in the predicting potential leachables and
in selecting the appropriate c/c system
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Leachables
 Migrate spontaneously from a c/c system and/or
other packaging components
normal conditions of use and storage
 Often a subset of extractables, or derived by
their chemical modification
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Sources of leachables in the product
 Syringes/prefilled syringes, ampoules, vials,
bottles
 IV bags
 Storage bags for product intermediates
 Closures (screw caps, rubber stoppers)
 Container liners (e.g., tube liners)
 Processing equipment:
• stainless steel storage tanks/bioreactors
• tubing
• gaskets, valves, rings
• filters
• purification resins
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Examples of leachables impacting on
safety and product quality
Example #1 – Impact of patient safety:
 Change: from HSA formulation to a polysorbate
 Unchanged container closure system (pre-filled syringes with the
uncoated rubber stoppers)
 Source: vulcanizing agents leached from rubber stopper over time
 Outcome:
• no detectable changes in product quality
• safety: serious adverse event (PRCA)
 Hypothesis: leachables acted as adjuvants triggering immunogenicity
Example #2 - Impact on product quality:
 Change from a lyophilized to a liquid formulation
 Divalent cation leached from the rubber stopper
 Caused activation of metalloprotease (a process-related impurity coeluted with the API)
 Impact: product degradation at the N-terminal site (stability study)
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Stability Program
 Drug Substance and Drug Product, real-time and
accelerated stability data with several time points under
upright and inverted conditions used to establish the
expiration period
 Stress studies (e.g., UV, exaggerated light, temperature
and pH) useful to elucidate product degradation pathways
and for defining acceptance criteria
 Limited time stability studies may be acceptable if shortterm trial is anticipated
 Stability data generated from engineering lots also
acceptable
 Failure to demonstrate product stability is a potential hold
issue
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Stability Program (cont.)
The following testing should be included at a minimum:
 Safety
• Bioburden/sterility
 Purity
• Product and process-related impurities & productrelated substances
 Sialic acid - if appropriate
 Potency
 Protein content/strength
 pH
 Appearance
 Leachables (separate study, not part of routine stability
testing)
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Good Manufacturing Practices
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Inspectional Activity
 Three types:
• Pre-licensed (PLI) - announced, generally required for approval
• Pre-approval (PAI) – announced, could be waived
• Surveillance (biennial post-licensure) – unannounced
• No formal inspection requirement for sites manufacturing
biologics under clinical investigation
• Manufacturing and testing sites are subject to inspection
 Inspection system undergoing revision for OBP products
 Currently inspections of facilities manufacturing CDER BLA products:
• PAI led by TFRB, Office of Compliance, with Product Reviewer(s)
sometimes part of on-site team
• Biennial post-licensure inspections led by Team Biologics with
Product Reviewers which can be part of the on-site team involved
in the inspection
 NDA Products:
• Pre-approval and post-licensure inspections led by district
personnel
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Facilities and Practices
 Closed systems
whenever possible
 Aseptic Processing
 CIP/SIP
 Disposable Systems
 Environmental
Monitoring
 Water/HVAC
 Good recordkeeping and
documentation
(phase 1)
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ICH Q7: Good Manufacturing Practice Guide
For Active Pharmaceutical Ingredients
Phase III
Phase I
Phase II
Provide greater assurance in linking product quality to commercial manufacture
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Potential Show Stoppers?
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Potential CMC Hold Issues
for Phase 1 IND
 Comparability between preclinical and clinical lots not
demonstrated
 Insufficient characterization of cell banks (e.g., adventitious
agents testing, identity, etc.)
 Inadequate product characterization with regards to purity,
identity, potency and safety
 Lack of final product release testing
 Lacking or inappropriate specifications for release and stability
testing
 Lacking or inadequate potency assay
 Data supporting product stability have not been shown for the
planned duration of clinical studies
 Lack or inappropriate immunogenicity assays for high risk
products
 Lack of evidence for final Drug Product sterility
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Guidance Documents
 Guidance for Industry: Content and Format of Investigational New Drug
Applications for Phase I Studies of Drugs, including Well-Characterized,
Therapeutic, Biotechnology derived Products (1995)
 Guidance for Industry for the Submission of CMC Information for a
Therapeutic Recombinant DNA-Derived Product or a Monoclonal Antibody
Product for In Vivo Use (1996)
 Guidance for Industry: IND for Phase 2 and 3 studies of Drugs, including
Specified Therapeutic Biotechnology-Derived Products – CMC Content and
Format (Draft, 1999)
 FDA Guidance Concerning Demonstration of Comparability of Human
Biological Products, including Therapeutic Biotechnology-derived Products
(1996)
 Guidance for Industry: INDs - Approaches to Complying with cGMP's for
Phase 1 Drugs (Draft, 2006)
 Points to Consider in the Manufacture and Testing of Monoclonal Antibody
Products for Human Use (1997)
 Points to Consider in the Characterization of Cell Lines Used to Produce
Biologicals (1993)
 International Conference on Harmonization (ICH) documents
 21 CFR 200’s, 600’s
 PHS Act, FD&C Act
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Acknowledgments
Emily Shacter
Barry Cherney
Steven Kozlowski
Wendy Shores
Susan Kirshner
Emanuela Lacana
Patricia Hughes
Joe Kutza
All of OBP
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Questions? Comments?
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